Bruce D. Meade

Use of Coefficient of Variation in Assessing Variability of Quantitative Assays

ABSTRACT We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation.

Establishment of Diagnostic Cutoff Points for Levels of Serum Antibodies to Pertussis Toxin, Filamentous Hemagglutinin, and Fimbriae in Adolescents and Adults in the United States

ABSTRACT Numerous reports have documented that serologic methods are much more sensitive than culture for the diagnosis of pertussis in adolescents and adults. However, a standardized serologic test for pertussis is not routinely available to most clinicians, and the serologic test levels or cutoff points correlated with diseases have not been determined. The goal of the present study was to examine the distribution of immunoglobulin G (IgG) levels against three Bordetella pertussis antigens (pertussis toxin [PT], filamentous hemagglutinin [FHA], and fimbria types 2 and 3 [FIM]) and to determine population-based antibody levels for the purpose of establishing such diagnostic cutoff points. Enzyme-linked immunosorbent assays (ELISAs) were performed with sera from >6,000 U.S. residents aged 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey. Mixture models were developed to identify hypothesized exposure groups and establish diagnostic cutoffs. Quantifiable (>20 ELISA units/ml [EU]) anti-FHA and anti-FIM IgG antibodies were common (65 and 62% of individuals, respectively), but quantifiable anti-PT IgG antibodies were less frequent (16%). Given the distributions of antibody levels, an anti-PT IgG level of ≥94 EU was proposed as the diagnostic cutoff point. Application of this cutoff point to culture-confirmed illness in a prior study investigating cough illness yielded a high diagnostic sensitivity (80%) and specificity (93%). A standardized ELISA for anti-PT IgG with a single serum sample appears to be useful for the identification of recent B. pertussis infection in adolescents and adults with cough illness. The PT cutoff point will be further evaluated in prospective studies of confirmed B. pertussis infection.

Safety and Immunogenicity of Six Acellular Pertussis Vaccines and One Whole-Cell Pertussis Vaccine Given as a Fifth Dose in Four- to Six-Year-Old Children

Objective. To evaluate the safety and immunogenicity of 6 different acellular pertussis vaccines combined with diphtheria and tetanus toxoids (DTaP) and with 1 licensed whole-cell pertussis vaccine (DTwP) as a fifth dose in children who had previously received the same DTaP, a different DTaP, or DTwP as primary and fourth-dose vaccinations. Methods. Healthy 4- to 6-year-old children were enrolled at 5 National Institute of Allergy and Infectious Diseases Vaccine Treatment and Evaluation Units to receive a fifth dose of a DTaP or DTwP vaccine. All had been randomly assigned to receive 3 primary doses of DTaP or DTwP at 2, 4, and 6 months and a fourth-dose booster at 15 to 20 months of age as part of earlier National Institutes of Health multicenter acellular pertussis vaccine trials. Parents recorded the occurrence and magnitude of fever, irritability, and injection site redness, swelling, and pain for 3 days after vaccination. Sera obtained before and 1 month after the booster vaccination were analyzed by enzyme-linked immunosorbent assay for antibody to pertussis toxin, filamentous hemagglutinin, fimbriae, pertactin, and diphtheria and tetanus toxoid. Safety and/or immunogenicity data are reported for 317 children who received DTaP and 10 children who received DTwP. Results. Fever and moderate or severe irritability were uncommon following the fifth dose of DTaP vaccine and were generally less frequent than following the fourth dose. However, for the DTaP vaccine groups, redness, swelling, and pain increased in prevalence compared with the fourth dose. The time course and frequency of reactions following DTaP vaccination were generally similar in children who received the same DTaP, a different DTaP, or DTwP for previous doses in the 5- dose series. No significant differences among the DTaP vaccines were detected in the occurrence of reactions, but the statistical power to detect differences was limited by sample size. Significant increases in antibodies directed against the included antigens were observed for all DTaP vaccines in paired pre- and post-fifth dose sera. Post-fifth dose antibody concentrations differed significantly among the DTaP vaccines. Some children in the study showed an antibody response to an antigen not reported to be in the DTaP vaccine. Conclusion. All the studied DTaP vaccines performed similarly with regard to reactions, whether given as a fifth sequential dose of the same vaccine, a mix of different DTaP vaccines in the 5-dose sequence, or after 3 DTwP and 1 DTaP vaccinations. Large injection site reactions occurred more frequently after the fifth dose of DTaP than after the previous 4 doses. A fifth dose of all DTaP vaccines induced an antibody response to those antigens contained in the vaccine. No DTaP was consistently most or least reactogenic or immunogenic.

Characterization of Reference Materials for Human Antiserum to Pertussis Antigens by an International Collaborative Study

ABSTRACT Enzyme-linked immunosorbent assay (ELISA) has been widely used to evaluate antibody responses to pertussis vaccination and infection. A common reference serum is essential for the standardization of these assays. However, no internationally recognized reference serum is available. At the request of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), a set of four candidate international standards has been prepared. These candidate materials have been assessed for suitability and compared to the widely used U.S. reference pertussis antiserum (human) lot 3, lot 4, and lot 5 by 22 laboratories from 15 countries in an international collaborative study. Laboratories measured immunoglobulin G (IgG) and IgA antibodies to pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (PRN), and fimbriae (Fim2&3) using their established immunoassays. The results of this study showed each of the four candidates to be suitable as an international standard. With the agreement of the participants, a recommendation has been made to the ECBS that the candidate material coded 06/140 be established as the First International Standard for pertussis antiserum (human), with the following assigned international units (IU): IgG anti-PT, 335 IU/ampoule; IgA anti-PT, 65 IU/ampoule; IgG anti-FHA, 130 IU/ampoule; IgA anti-FHA, 65 IU/ampoule; IgG anti-PRN, 65 IU/ampoule; and IgA anti-PRN, 42 IU/ampoule. No formal units have been proposed for anti-Fim2&3 because most assays used a mixture of fimbrial antigens. In addition, the candidate material coded 06/142 has been proposed as a WHO working preparation for characterization of assay systems.

Pertussis Resurgence: Perspectives From the Working Group Meeting on Pertussis on the Causes, Possible Paths Forward, and Gaps in Our Knowledge

The incidence of reported pertussis has steadily increased in the United States since the 1980s, with a striking increase observed since 2005 [1]. Provisional case counts in the United States for 2012 exceed 41 000, with 14 deaths among infants [2]. This resurgence in pertussis that has been seen in the United States and other countries [3] has caused considerable concern. On 6 March 2013, 64 experts in the field of pertussis vaccinology, from academia, government agencies, and pharmaceutical companies, met during the Working Group Meeting on Pertussis that was held in Bethesda, Maryland. This workshop offered a unique opportunity for experts to discuss possible causes of the resurgence in pertussis, the possible paths forward, and the remaining gaps in our knowledge. To take advantage of this collection of experts, 3 discussion sessions were held during the workshop to discuss (1) potential root cause(s) of the problem, (2) what interim solutions may exist, and (3) how the problem might be solved in the long term. At the end of the workshop, the experts agreed that use of the currently available vaccines is an important strategy but that longer-term solutions are necessary to more effectively control pertussis. No clear consensus was reached as to a single root cause of the problem nor did a clear long-term solution emerge. However, the group began to define gaps in knowledge, discussed hypotheses that could be tested, and suggested priorities for research and development.

Structural and immunological analysis of anthrax recombinant protective antigen adsorbed to aluminum hydroxide adjuvant.

ABSTRACT New anthrax vaccines currently under development are based on recombinant protective antigen (rPA) and formulated with aluminum adjuvant. Because long-term stability is a desired characteristic of these vaccines, an understanding of the effects of adsorption to aluminum adjuvants on the structure of rPA is important. Using both biophysical and immunological techniques, we compared the structure and immunogenicity of freshly prepared rPA-Alhydrogel formulations to that of formulations stored for 3 weeks at either room temperature or 37°C in order to assess the changes in rPA structure that might occur upon long-term storage on aluminum adjuvant. Intrinsic fluorescence emission spectra of tryptophan residues indicated that some tertiary structure alterations of rPA occurred during storage on Alhydrogel. Using anti-PA monoclonal antibodies to probe specific regions of the adsorbed rPA molecule, we found that two monoclonal antibodies that recognize epitopes located in domain 1 of PA exhibited greater reactivity to the stored formulations than to freshly prepared formulations. Immunogenicity of rPA-Alhydrogel formulations in mice was assessed by measuring the induction of toxin-neutralizing antibodies, as well as antibodies reactive to 12-mer peptides spanning the length of PA. Mice immunized with freshly prepared formulations developed significantly higher toxin-neutralizing antibody titers than mice immunized with the stored preparations. In contrast, sera from mice immunized with stored preparations exhibited increased reactivity to nine 12-mer peptides corresponding to sequences located throughout the rPA molecule. These results demonstrate that storage of rPA-Alhydrogel formulations can lead to structural alteration of the protein and loss of the ability to elicit toxin-neutralizing antibodies.

Development and Analytical Validation of an Immunoassay for Quantifying Serum Anti-Pertussis Toxin Antibodies Resulting from Bordetella pertussis Infection

ABSTRACT Adequately sensitive and specific methods to diagnose pertussis in adolescents and adults are not widely available. Currently, no Food and Drug Administration-approved diagnostic assays are available for the serodiagnosis of Bordetella pertussis. Since concentrations of B. pertussis-specific antibodies tend to be high during the later phases of disease, a simple, rapid, easily transferable serodiagnostic test was developed. This article describes test development, initial evaluation of a prototype kit enzyme-linked immunosorbent assay (ELISA) in an interlaboratory collaborative study, and analytical validation. The data presented here demonstrate that the kit met all prespecified criteria for precision, linearity, and accuracy for samples with anti-pertussis toxin (PT) immunoglobulin G (IgG) antibody concentrations in the range of 50 to 150 ELISA units (EU)/ml, the range believed to be most relevant for serodiagnosis. The assay met the precision and linearity criteria for a wider range, namely, from 50 to 200 EU/ml; however, the accuracy criterion was not met at 200 EU/ml. When the newly adopted World Health Organization International Standard for pertussis antiserum (human) reference reagent was used to evaluate accuracy, the accuracy criteria were met from 50 to 200 international units/ml. In conclusion, the IgG anti-PT ELISA met all assay validation parameters within the range considered most relevant for serodiagnosis. This ELISA was developed and analytically validated as a user-friendly kit that can be used in both qualitative and quantitative formats. The technology for producing the kit is transferable to public health laboratories.

International Bordetella pertussis assay standardization and harmonization meeting report. Centers for Disease Control and Prevention, Atlanta, Georgia, United States, 19–20 July 2007

Abstract An international meeting on Bordetella pertussis assay standardization and harmonization was held at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, 19–20 July 2007. The goal of the meeting was to harmonize the immunoassays used for pertussis diagnostics and vaccine evaluation, as agreed upon by academic and government researchers, regulatory authorities, vaccine manufacturers, and the World Health Organization (WHO). The primary objectives were (1) to provide epidemiologic, laboratory, and statistical background for support of global harmonization; (2) to overview the current status of global epidemiology, pathogenesis and immunology of pertussis; (3) to develop a consensus opinion on existing gaps in understanding standardization of pertussis assays used for serodiagnosis and vaccine evaluation; and (4) to search for a multicenter process for addressing these priority gaps. Presentations and discussions by content experts addressed these objectives. A prioritized list of action items to improve standardization and harmonization of pertussis assays was identified during a group discussion at the end of the meeting. The major items included: (1) to identify a group that will organize, prepare, maintain, and distribute proficiency panels and key reagents such as reference and control sera; (2) to encourage the development and identification of one or more reference laboratories that can serve as an anchor and resource for other laboratories; (3) to define a performance-based assay method that can serve as a reference point for evaluating laboratory differences; (4) to develop guidance on quality of other reagents, e.g., pertussis toxin and other antigens, and methods to demonstrate their suitability; (5) to establish an international working group to harmonize the criteria to evaluate the results obtained on reference and proficiency panel sera; (6) to create an inventory to determine the amount of appropriate and well-characterized sera that are available globally to be used as bridging reagents for vaccine licensure; and (7) to seek specific guidance from regulatory authorities regarding the expectations and requirements for the licensure of new multicomponent pertussis vaccines.

Pertussis in a previously immunized child with human immunodeficiency virus infection

The management of children with human immunodeficiency virus infection in whom changes in pulmonary status develop can be complex. They are susceptible to a broad range of infections, and the clinician must be aware of the multiple etiologic possibilities and appropriate diagnostic tests. This report presents a potential pathogen that should be added to the list of organisms that can cause gram-negative pneumonia in children with HIV infection and highlights a diagnostic pitfall.

Analysis of the Fc Gamma Receptor-Dependent Component of Neutralization Measured by Anthrax Toxin Neutralization Assays

ABSTRACT Anthrax toxin neutralization assays are used to measure functional antibody levels elicited by anthrax vaccines in both preclinical and clinical studies. In this study, we investigated the magnitude and molecular nature of Fc gamma (Fcγ) receptor-dependent toxin neutralization observed in commonly used forms of the anthrax toxin neutralization assay. Significantly more Fcγ receptor-dependent neutralization was observed in the J774A.1 cell-based assay than in the RAW 264.7 cell-based assay, a finding that could be due to the larger numbers of Fcγ receptors that we found on J774A.1 cells by using flow cytometry. Thus, the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by the assay depends on the specific cell type utilized in the assay. Using Fcγ receptor blocking monoclonal antibodies, we found that at least three murine Fcγ receptor classes, IIB, III, and IV, can contribute to Fcγ receptor-dependent neutralization. When antibodies elicited by immunization of rabbits with protective-antigen-based anthrax vaccines were analyzed, we found that the magnitude of Fcγ receptor-dependent neutralization observed in the J774A.1 cell-based assay was dependent on the concentration of protective antigen utilized in the assay. Our results suggest that the characteristics of the antibodies analyzed in the assay (e.g., species of origin, isotype, and subclass), as well as the assay design (e.g., cell type and protective antigen concentration), could significantly influence the extent to which Fcγ receptor-dependent neutralization contributes to the total neutralization measured by anthrax toxin neutralization assays. These findings should be considered when interpreting anthrax toxin neutralization assay output.